The triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were examined for their inhibitory effect on highly purified cellular DNA polymerase alpha and human cytomegalovirus (Towne strain)-induced DNA polymerase. These two nucleoside triphosphates competitively inhibited the incorporation of dGMP into DNA catalyzed by the DNA polymerases. The virus-induced DNA polymerase had greater binding affinity for the triphosphate of 9-(2-hydroxyethoxymethyl)guanine (Ki, 8 nM) than for the triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (Ki, 22 nM), although the nucleoside of the latter compound was strikingly more effective against human cytomegalovirus replication in cell cultures than the nucleoside of the former. The Ki values of these two nucleoside triphosphates for alpha polymerase were 96 and 146 nM, respectively, and were 7- to 12-fold higher than those for the virus-induced enzyme. These data indicated that virus-induced DNA polymerase was more sensitive to inhibition by these two nucleoside triphosphates than was the cellular alpha enzyme.